JCM Accepted Manuscript Posted Online 27 April 2020

J. Clin. Microbiol. doi:10.1128/JCM.00708-20

Copyright © 2020 Rogers et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

1 Evaluation of Transport Media and Specimen Transport Conditions for the Detection of SARS-CoV-2
2 Using Real Time Reverse Transcription PCR

3 Amy A. Rogers1, Russell E. Baumann1, Gwynngelle A. Borillo1, Ron M. Kagan 1, Hollis J. Batterman1,
4 Marzena Galdzicka2 and Elizabeth M. Marlowe 1
1
5 Quest Diagnostics Infectious Disease, San Juan Capistrano, CA USA
2
6 Quest Diagnostics Marlborough, MA USA

7 Submitted: April 8, 2020

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8 Revised: April 22, 2020
9 In Preparation for Submission to JCM as a Research Article

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19 Corresponding Author:

20 Elizabeth M. Marlowe, PhD, D(ABMM), SM(ASCP)

21 Scientific Director, Head R&D West, Infectious Disease
22 Quest Diagnostic Infectious Disease
23 33608 Ortega Highway
24 San Juan Capistrano, CA 92675-2042
25 email: Elizabeth.m.marlowe@questdiagnostics.com
26 phone 949.728.1817
27 fax 949.728-7180
28
29 Abstract: The global COVID-19 pandemic has resulted in a worldwide shortage of viral transport media
30 and raised questions about specimen stability. The objective of this study was to determine the stability
31 of SARS-CoV-2 virus RNA in specimen transport media under various storage conditions. Transport
32 medium tested included: VCM, UTM®-RT, ESwab™, M4 and saline (0.9% NaCl). Specimen types tested
33 included Nasopharyngeal/Oropharyngeal (NP/OP) swabs in the above transport media, bronchoalveolar
34 lavage (BAL) and Sputum. A high-titer SARS-CoV-2 remnant patient specimen was spiked into pooled
35 SARS-CoV-2 RNA-negative specimen remnants for the various media types. Aliquots of samples were
36 stored at 18°C to 25°C, 2°C to 8°C and -10°C to -30°C and then tested at time points up to 14 days.
37 Specimens consistently yielded amplifiable RNA with mean Ct differences of <3 over the various

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38 conditions assayed, thus supporting the use and transport of alternative collection media and specimen
39 types under a variety of temperature storage conditions.

40 Introduction:

41 On December 31, 2019, an outbreak of respiratory disease caused by a novel coronavirus first
42 detected in Wuhan City, Hubei Province, China was initially reported to the World Health Organization
43 (WHO) and has continued to expand globally(1, 2). On January 30, 2020 the United States reported the
44 first confirmed instance of person-to-person spread of SARS-CoV-2, to an individual who had close
45 contact to a known case(2). On March 11, 2020 the WHO declared COVID-19 a pandemic(3).

46 Coronaviruses are a large family of viruses that are common in many different species, including camels,
47 cattle, cats, and bats(2). Globally, there are four common human Coronaviruses in circulation; 229E,
48 NL63, OC43, and HKU1. Infection and spread of animal coronaviruses into people is rare. Cases of
49 zoonotic transmission of animal coronaviruses include MERS-CoV, SARS-CoV-1, and SARS-CoV-2
50 (previously known as 2019-novel coronavirus, 2019-nCoV) have been described (2). Since the first
51 reported cases of SARS-CoV-2 in December 2019 greater the 2.6 million cases have been reported
52 globally as of April 22, 2020, according to the COVID-19 dashboard by the Center for Systems Science
53 and Engineering at John Hopkins University
54 (https://www.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6).

55 The COVID-19 pandemic has resulted in an unprecedented worldwide demand for laboratory testing.
56 The huge increase in testing has put pressure on the laboratory supply chain and resulted in shortages of
57 viral transport media. The Food and Drug Administration (FDA) has recommended the use of alternative
58 viral transport media, such as liquid amies, saline and phosphate buffered saline. Further
59 recommendations suggest specimens can be stored for up to 72 hours at 4℃, or frozen at ≤ -70℃ for
60 longer storage(4). The objective of this study was to evaluate the detection of SARS-CoV-2 RNA by rRT-
61 PCR, using pooled remnant respiratory specimens placed into different transport media and held under
62 common specimen transport conditions.

63 Materials and Methods:

64 Clinical Specimens: Transport media tested at Quest Diagnostics Infectious Disease (QDID, San Juan
65 Capistrano, CA) included VCM (Copan, Brescia, Italy), UTM®-RT (Copan, Brescia, Italy), ESwab™(Copan,
66 Brescia, Italy), M4 media (Thermo Fisher Scientific, Waltham, MA) and Normal Saline [0.9% NaCl].
67 Specimen types tested included NP/OP swabs in the various transport media, bronchoalveolar lavage
68 (BAL) and sputum. Prior to spiking, remnant sputum had been processed in 1X PBS. A known high titer
69 SARS-CoV-2 positive specimen was utilized to spike SARS-CoV-2 RNA-negative specimen remnants for
 70 the various media. The RNA viral load was estimated based on cycle threshold (Ct) values to be
71 approximately 1,500 copies/mL.

72 Study Design: Samples were aliquoted and stored for up to 14 days and tested at multiple time points
73 and storage temperatures (18°C to 25°C, 2°C to 8°C and -10°C to -30°C). Five aliquots were assayed at
74 each condition. Molecular analysis was performed utilizing the QDID SARS-CoV-2 RNA, Qualitative Real-
75 Time RT-PCR EUA assay according the package instructions for use (5). A positive result for SARS-CoV-2
76 RNA was defined as a Ct value of <40 for both the N1 and N3 detectors. Samples were tested before
77 storage (T=0) to obtain an initial Ct result. Samples were deemed stable if the mean Ct values were

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78 within ≤ 3 amplification cycles of the mean initial Ct value.

79 Additional Saline Studies: Storage studies using Normal Saline [0.9% NaCl] were also performed at
80 Quest Diagnostics Marlborough, MA, using the Quest Diagnostics and the Roche Diagnostics cobas®
81 SARS-CoV-2 EUA tests. A high-titer SARS-CoV-2 positive patient specimen with a Ct of 18 established the
82 Roche cobas® system was diluted 1:1,000 to obtain a calculated Ct value of 28. For each time point,
83 samples were tested in triplicate at this concentration. A further 1:10 dilution of this material
84 (calculated Ct = 31) was also tested in duplicate at each time point.

85 Statistical Analysis: Statistical analysis was performed using Analyse-it for Microsoft Excel version
86 5.40.2.

87 Results:

88 SARS-CoV-2 RNA was consistently detected in all transport media, specimen types, and storage
89 conditions tested; mean Ct values obtained for SARS-Cov-2 for the various study-defined transport
90 media and storage temperatures are shown in Table 1. Differences in average viral RNA Ct values were
91 similar across all media and temperature storage conditions assayed (Table 1). Mean Ct differences
92 between Day 0 and Day 7 for all media tested at room temperature were 0.6 ± 0.7 Cts. Refrigerated and
93 frozen samples exhibited mean Ct differences of 0.5 ± 0.7 and 0.7 ± 1.1 Cts respectively. For frozen
94 ESwab™ samples one of five replicates at day 5 did not yield detectable RNA for the N3 target (Table 1).
95 Detection of only one of two targets in the assay is considered an inconclusive result. For saline and
96 ESwab™ transport media there was a shift of >2 Cts in the average Ct between Day 0 and Day 7 and/or
97 Day 14 (Table 1 & 2). These changes would not have altered the interpretation of positive results.

98 A further stability study performed at a second site assessed the stability of SARS-CoV-2 RNA in
99 saline for up to 14 days using both the Quest and the Roche cobas® EUA assays. As shown in Table 2,
100 SARS-Cov-2 RNA remained detectable for 14 days on both platforms. For the samples tested by the
101 Quest and the cobas® EUA on frozen saline, we observed minimal variation (<1 Ct on average) in the
102 mean Ct values over 14 days. However, at room temperature and refrigerated storage conditions, we
103 noted a >2 Ct increase over 14 days (Table 2). The increase in the Ct values was linear over 14 days, with
104 a slope of 0.14 and 0.15 Cts per day for the two targets (R2: 0.79 and 0.78) at room temperature and
105 0.13 Cts per day (R2: 0.83 and 0.81) while stored refrigerated. A further 1:10 dilution of the SAR-CoV-2
106 RNA (calculated Ct = 31) was also tested in duplicate at each time point and comparable results and
107 trends were observed (data not shown).

108

109 Discussion:
110 Limited stability studies are available in the literature for SARS-CoV-2 RNA. A study of SARS-CoV-2
111 in aerosols and on surfaces demonstrated that culturable SAR-CoV-2 was detectable in aerosols for up
112 to three hours, up to four hours on copper, up to 24 hours on cardboard and up to two to three days on
113 plastic and stainless steel (6). Given the persistence of SARS-CoV-2 in the environment, it is not
114 surprising that RNA can be reliably amplified from viral transport media after relatively long storage
115 times, even at room temperature.

116 Prior stability studies of SARS-Cov-2 RNA in various transport media and conditions are also limited. The
117 results reported here are consistent with the findings of Druce et al (7) who examined the stability of

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118 four common viruses with different physicochemical properties in several swab and transport media and
119 storage combinations. The authors demonstrated that influenza, enterovirus, herpes simplex virus and
120 adenovirus were detected by PCR at 22°C and refrigerated 4°C for up to 7 days.

121 Stability using various viral transport media performed with in-house laboratory developed tests for
122 Influenza and Rubeola viruses (both enveloped RNA viruses) are also consistent with viral RNA detection
123 at 18 to 26°C after 7 days, 2 to 8°C after 14 days and -10 to -30°C after 30 days (data not shown).
124 Rodino et al (8) demonstrated reliable detection of SARS-CoV-2 RNA in swab stored in MEM, PBS, Saline
125 and VTM after 7 days at 2-8°C and frozen at -20°C using an in-house EUA as well as the Roche cobas®
126 EUA.

127 Qualitative detection of SARS-CoV-2 RNA was unchanged over the various combinations of transport
128 media and conditions tested at two study sites regardless of molecular platform utilized. While stability
129 in saline stored at room temperature and under refrigerated conditions exhibited a linear trend with
130 increasing Ct values over time, these trends did not impact the qualitative interpretation of positive
131 results. Additionally, specimen stability was assessed in this study for a significantly longer duration
132 than would be deemed acceptable for routine clinical testing, further reducing the probability of clinical
133 impact. The greatest theoretical impact of this linear trend would be for specimens with low titers of
134 virus. In our experience with clinical specimens, however, the majority of positive specimens tested
135 exhibited low Ct values (<30) which correlate to specimens with higher titers of virus. Viral titers may be
136 impacted by the clinical course at time of sample acquisition and the quality of the specimen collection.
137 These data provide additional supporting evidence for the use of alternative viral transport media and
138 temperature storage conditions for the detection SARS-CoV-2 RNA using sensitive rRT-PCR assays.
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139 Table 1. Stability of SARS-COV-2 RNA detected by the Quest EUA rRT-PCR1
140
141
Room Temperature (18 to 26°C)
N=5; mean (sd)
Media/sample type Detector2 Day 0 Day 2 Day 3 Day 5 Day 7
N1 31.8 (0.2) 31.6 (0.4) 31.6 (0.3) 31.6 (0.4) 31.5 (0.8)
VCM
N3 31.3 (0.4) 30.8 (0.2) 30.8 (0.5) 30.8 (0.4) 30.7 (0.7)
N1 31.8 (0.3) 31.6 (0.3) 31.3 (0.2) 31.9 (0.4) 33.1 (0.4)
UTM®-R
N3 31.2 (0.4) 30.8 (0.3) 31.0 (0.2) 31.1 (0.3) 32.2 (0.4)
N1 31.7 (0.4) 32.0 (0.4) 31.9 (0.5) 31.9 (0.4) 32.6 (1.0)
ESwab™
N3 31.3 (0.3) 31.1 (0.2) 30.9 (0.3) 31.0 (0.3) 31.6 (0.7)
N1 31.8 (0.2) 32.1 (0.4) 31.5 (0.2) 32.4 (0.5) 32.6 (0.6)
M4
N3 31.3 (0.4) 31.2 (0.4) 31.0 (0.1) 31.2 (0.2) 31.6 (0.4)
N1 29.2 (0.7) 29.9 (0.3) 30.3 (0.1) 30.7 (0.3) 31.1 (0.3)
Saline
N3 28.4 (0.6) 29.1 (0.2) 29.5 (0.1) 29.9 (0.5) 30.0 (0.2)
N1 31.8 (0.2) 31.5 (0.2) 32.2 (0.5) 31.4 (0.3) 32.4 (0.4)
BAL
N3 31.1 (0.2) 30.8 (0.2) 31.2 (0.4) 30.6 (0.3) 31.2 (0.4)
N1 31.4 (0.5) 31.8 (0.6) 32.1 (0.4) 31.8 (0.4) 32.1 (0.3)
Sputum
N3 30.8 (0.6) 31.1 (0.7) 31.1 (0.5) 30.8 (0.3) 31.1 (0.3)
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144

Refrigerated (2 to 8°C)
N=5; mean (sd)
Media/sample type Detector2 Day 0 Day 3 Day 5 Day 7 Day 10 Day 14
N1 31.8 (0.2) 31.8 (0.2) 31.8 (0.4) 31.9 (0.6) 32.2 (0.4) 32.1 (0.3)
VCM
N3 31.3 (0.4) 31.3 (0.4) 31.1 (0.5) 31.0 (0.5) 31.6 (0.4) 31.4 (0.4)
N1 31.8 (0.3) 31.4 (0.3) 32.1 (0.1) 31.1 (1.8) 32.5 (0.2) 32.1 (0.3)
UTM®-R
N3 31.2 (0.4) 30.8 (0.4) 31.3 (0.2) 30.4 (1.7) 31.6 (0.1) 31.4 (0.3)
N1 31.7 (0.4) 31.9 (0.3) 31.7 (0.2) 31.8 (0.3) 31.7 (0.4) 31.8 (0.4)
ESwab™
N3 31.3 (0.3) 31.0 (0.4) 30.7 (0.2) 30.7 (0.3) 30.8 (0.4) 31.2 (0.4)
N1 31.8 (0.2) 31.7 (0.3) 32.0 (0.4) 31.5 (1.5) 31.8 (0.5) 31.9 (0.2)
M4
N3 31.3 (0.4) 31.0 (0.3) 31.1 (0.3) 30.7 (1.2) 31.3 (0.6) 31.3 (0.3)
N1 29.2 (0.7) 30.1 (0.2) 30.0 (0.4) 30.1 (1.7) 30.4 (1.2) 31.3 (0.4)3
Saline
N3 28.4 (0.6) 28.9 (0.2) 29.0 (0.5) 28.9 (1.4) 29.4 (1.0) 30.5 (0.4)3
N1 31.8 (0.2) 32.2 (0.6) 31.0 (1.4) 32.5 (0.1) 32.1 (0.6) 32.4 (0.7)
BAL
N3 31.8 (0.2) 32.2 (0.6) 31.0 (1.4) 32.5 (0.1) 32.1 (0.6) 31.5 (0.5)
N1 31.4 (0.5) 31.4 (0.5) 31.8 (0.6) 32.1 (0.5) 32.1 (0.4) 32.2 (0.7)
Sputum
N3 30.8 (0.6) 30.7 (0.9) 31.0 (0.4) 31.0 (0.4) 31.8 (0.6) 31.6 (0.8)
145

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151
Frozen (-10-to -20°C)
N=5; mean (sd)
Media/sample type Detector3 Day 0 Day 3 Day 5 Day 7 Day 14
N1 31.8 (0.2) 31.9 (0.4) 31.5 (0.3) 32.0 (0.4) 32.0 (0.4)
VCM
N3 31.3 (0.4) 31.1 (0.3) 30.6 (0.4) 31.1 (0.3) 31.2 (0.4)
N1 31.8 (0.3) 31.7 (0.4) 31.9 (0.4) 32.3 (0.2) 31.7 (0.4)
UTM®-R
N3 31.2 (0.4) 31.2 (0.3) 31.0 (0.4) 31.4 (0.4) 31.0 (0.2)
N1 31.7 (0.4) 34.3 (1.1) 34.8 (0.4) 31.8 (0.3) 33.5 (0.6)
ESwab™
N3 31.3 (0.3) 33.7 (0.9) 34.5 (0.4)* 31.7 (0.4) 33.8 (0.6)3
N1 31.8 (0.2) 32.1 (0.7) 32.0 (0.5) 31.0 (3.0) 31.5 (0.2)
M4
N3 31.3 (0.4) 31.6 (0.7) 31.1 (0.5) 30.1 (3.0) 31.0 (0.3)
N1 29.2 (0.7) 30.6 (0.5) 31.4 (0.3) 31.7 (0.4)3 31.6 (0.9)3
Saline
N3 28.4 (0.6) 29.6 (0.4) 30.3 (0.3) 30.6 (0.4)3 30.7 (0.9)3
N1 31.8 (0.2) 32.4 (0.6) 32.2 (0.4) 32.5 (0.3) 32.5 (0.1)
BAL
N3 31.1 (0.2) 31.3 (0.4) 31.3 (0.3) 31.8 (0.3) 31.6 (0.2)
N1 31.4 (0.5) 31.8 (1.0) 31.6 (0.3) 31.8 (0.5) 31.8 (0.3)
Sputum
N3 30.8 (0.6) 30.9 (1.0) 30.7 (0.1) 30.8 (0.5) 31.0 (0.4)
* 1/5 not detected
152
153
154
1
155 Testing was performed at QDID. Five replicates were assayed by rRT-PCR at each of the indicated conditions
2
156 N1 and N3 Taqman® probes as described in the Quest Diagnostics EUA package insert
3
157 Increase of >2 Cts from Day 0
158
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159 Table 2. Additional Stability of SARS-COV-2 RNA in Saline detected by by the Quest EUA and Roche cobas EUA1

160

Room Temperature (18 to 26°C)

N=3; mean (sd)
Platform Detector2 Day 0 Day 1 Day 2 Day 3 Day 5 Day 7 Day 14
N1 26.2 (0.1) 26.3 (0.0) 26.1 (0.5) 26.8 (0.1) 27.2 (0.1) 26.4 (0.1) 26.8 (0.4)
Quest EUA
N3 30.2 (0.1) 30.3 (0.1) 30.0 (0.4) 30.7 (0.1) 31.5 (0.1) 29.3 (0.1) 29.3 (0.3)
ORF 1a/b 27.9 (0.3) 28.4 (0.2) 28.5 (0.1) 29.1 (0.1) 29.4 (0.2) 29.5 (0.0) 30.0 (0.2)3
Cobas
E gene 28.1 (0.3) 28.6 (0.2) 28.7 (0.1) 29.3 (0.2) 29.7 (0.2) 29.9 (0.2) 30.4 (0.2)3
161

Refrigerated (2 to 8°C)
N=3; mean (sd)
Platform Detector2 Day 0 Day 1 Day 2 Day 3 Day 5 Day 7 Day 14
N1 26.2 (0.1) 26.4 (0.1) 26.0 (0.2) 26.5 (0.0) 27.0 (0.1) 26.3 (0.2) 26.5 (0.2)
Quest EUA
N3 30.2 (0.1) 30.2 (0.1) 29.8 (0.2) 30.6 (0.0) 31.3 (0.1) 29.0 (0.2) 29.1 (0.1)
ORF 1a/b 27.9 (0.3) 28.5 (0.1) 28.5 (0.1) 28.8 (0.1) 29.2 (0.1) 29.3 (0.1) 29.9 (0.1)
Cobas
E gene 28.1 (0.3) 28.8 (0.1) 28.7 (0.1) 29.1 (0.1) 29.5 (0.1) 29.5 (0.2) 30.2 (0.2)3
162

Frozen (-70 to -90°C)

N=3; mean (sd)
Platform Detector2 Day 0 Day 1 Day 2 Day 3 Day 5 Day 7 Day 14
N1 26.2 (0.1) 26.3 (0.0) 26.1 (0.5) 26.8 (0.1) 27.2 (0.1) 26.4 (0.1) 26.8 (0.4)
Quest EUA
N3 30.2 (0.1) 30.3 (0.1) 30.0 (0.4) 30.7 (0.1) 31.5 (0.1) 29.3 (0.1) 29.3 (0.3)
ORF 1a/b 27.9 (0.3) 28.4 (0.1) 28.2 (0.2) 28.7 (0.2) 28.5 (0.2) 28.3 (0.1) 28.4 (0.1)
Cobas
E gene 28.1 (0.3) 28.7 (0.2) 28.4 (0.3) 28.7 (0.1) 28.8 (0.1) 28.6 (0.1) 28.7 (0.0)
163
164
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1
165 Testing was performed at Quest Diagnostics Marlborough, MA. Three replicates were assayed by rRT-PCR at each of the indicated conditions
2
166 N1 and N3 Taqman® probes as described in the Quest Diagnostics EUA package insert, and ORF 1a/1b and E gene probes as described in the
167 Roche Diagnostics EUA package insert
3
168 Increase of >2 Cts from Day 0
169

170 References

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